Bacterial classification Wall structure

Gram + Staphylococcus, Streptococcus,

Clostridium, Bacillus Gram -

Enteric, respiratory and others Acid-fast

Mycobacterium Wall-less

Mycoplasma Unusual

Obligate intracellular Rickettsia, Chlamydia, Treponema

G+ G- AF WL IC

Bacteria

Bacterial classification

Cell morphology Shapes

Rod Cocci Spiral

Associations Individual Diplo- Staphylo- Strepto- Filaments

G+ G- AF WL IC

Bacteria

Rod Cocci Rod Cocci Spiral

Gram stains

Gram Stains of Mixtures

Cell shapes

Bacterial classification

Growth characteristics Oxygen requirement

AerobicAnaerobic

Microaerophilic, aerotolerantFacultative

Spore formation Intracelular/extracellular Fastidious/non-fastidious

Endospore

Classification & Diagnosis

Type of colonies Appearance

Color, shape, size and smoothness On differential media

Blood, MacConkey, EMB, Manitol salt agar

On selective mediaMacConkey, EMB, Manitol salt agar

Colony morphology/color

Bacterial colony morphology

Capsule

MacConkey Agar Plate

Blood Agar Plates and Hemolysis

Mannitol salt agar

Classification & Diagnosis Metabolism

Utilization of specific substrates Lactose (Sal/Shi/Yer/)-

Citrate (E.coli-/Klebsiela+)

Production of certain end products Fermentation end products

Acid (acetate, propionic acid, butyric acid etc.)

Aceton Alcohol Amine H2S

Triple Sugar Iron Agar Slants

TSI•Fermentation of glucose, lactose, and/or sucrose•Reduction of sulfur to hydrogen sulfide•Gas formation

Used for Enterobacteriaceae

Classification & Diagnosis Specialized tests

Immunological O-, H- & K-Ag (serotype) Precipitation, agglutination

Specialized enzymes Catalase--- Staph+. vs. Strep-. Coagulase---S. aureus+ vs. S.

epidermidis-

Oxidase---Neisseria gonorrhoea+

Urease---Proteus+, Helicobacter+

Antibiogram pattern Phage typing Fatty acid profile

Urease Test

Urea CO2 + NH3 NH4+ + OH-

H2O

Indole Test

API test strip

Bacteriophage Plaques

Conventional method

Depend on ability to culture Treponema pallidum

Slow, esp. for fastidious species Mycobacterium spp.

Not always definitive

Immunological detection

ELISA

Instant View tests

Molecular diagnosis

Ribotyping Restriction fragment length

polymorphism (RFLP) DNA hybridization PCR, RT-PCR and RAPD Nucleic acid sequence

analysis Phage-GFP (TB)

RFLP

GGATCCCCTAGG

DNA hybridization

In situHybridization

Rate of increase2n

PCR

Real time PCRQR

5'3' 5'

3'

5' 3' 3' 5'

QR

5' 3' 3' 5'

QR

Q

5' 3' 3' 5'

R

PrimPrimerer

PrimePrimerr

ProbeProbe

Taq DNA Taq DNA PolPol

RAPD of P. aeruginosa

DNA and protein chips

A B C D

Microarray array mediated pathogen detection

Ab chip mediated pathogen detection

-O157:H7

-Salmonella

-O157:H7

-Salmonella

E.coli O157:H7

Salmonellatyphimurium

Molecular diagnosis

Reduce reliance on culture

Faster High sensitive More definitive More discriminating Techniques

adaptable to all pathogens

Technically demanding

Relatively expensive

Can be too sensitive

Provides no information if results are negative

Bioterrorism

Pathogen detection Fast and accurateMobile Inexpensive

Source investigation

Biosensor Mediated Pathogen Detection

Step 1: Attach Capture Antibody to Fiber Optic Waveguide

Step 2: Apply Sample. Target Organism Binds to WaveguideStep 3: Wash Away Other Cells and Debris

Y Y Y Y YY YY Y YY YY

YY YYYY

Step 1: Attach Capture Antibody to Fiber Optic WaveguideStep 2: Apply Sample. Target Organism Binds to WaveguideStep 3: Wash Away Other Cells and DebrisStep 4: Add Specific Detection Antibody Step 5: Turn on Laser. Detection Antibody Fluoresces,

Indicating a Positive Sample.

Y Y Y Y YY YY Y YYY

Y YYYY

Y Y Y YY YY YY

Y Y YY Y YY Y

Y Y

Biosensor Mediated Pathogen Detection

Biosensors

Analyte 2000TM

for laboratory research and development

RAPTORTM automated version for field

analysis

Differentiating Staphylococci from Streptococci

Gram stain and morphology Both Gram + Staphylococci: bunched cocci Streptococci: chained cocci

S. pneumoniae: diplococcus

Enzyme tests Staphylococci: catalase + Streptococci: catalase -

Growth Staph.: large colonies (non-fastidious), some hemolytic Strep.: small colonies (fastidious), many hemolytic (

or )

Staphylococci

S. aureus : coagulase + S. epidermidis : coagulase -

Streptococci

Streptococci On blood agar Growth inhibition discS. pyogenes (group A) -hemolytic Sensitive to bacitracinS. agalactiae (group B) -hemolytic Resistant to bacitracin

S. pneumoniae (pneumococcus) -hemolytic Sensitive to optochinViridans -hemolytic Resistant to optochin

Differentiating the Gram- bacteria

Cocci Neisseria

Rods Type of disease they cause Enteric Gram- rods

API test Curved

Vibrio, Campylobacter, Helicobacter

Spiral Gram- organisms Spirochetes

Gram negative

Straight rods Curved rods

Lactose+ Lactose-

Citrate+ Citrate- H2S+ H2S-

Klebsiella E.coli Salmonella Shigella

Campy blood agar42oC+ 25oC-

Campylobacter

TCBS agarYellowOxidase+

Vibrio

BacteriaGram+

Cocci Rod

Staph. Strep. Non-spore Spore

S. a.S. e.S. s.

ABPnVir

Fil Rod

A.i. C.d.L. m.

+O2 -O2

B.a.B.c.

C.b.C.t.C.p.C.d.

Acid Fast

M.t.M.l.N.c.

IntraCellular

RickettsiaCoxiellaErlichiaChlamydia

WallLess

Mycoplasma

Gram-

CocciRodSpiral

StraightCurve

+O2 -O2+/-O2 Other

TreponemaBorreliaLeptospira

NeisseriaMoraxella

P.a. Enteric Bact.

Resp. Zoo GU

Bordetella.H. influenzaeLegionella

YersiniaPasteurellaBrucellaFrancisllaStreptobacillus

H. ducreyiGardnerellaCalymmatobacterium

VibrioCampylobacterHelicobacter